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M. Bitzer, N.C. Brecha; Expression of Different VAMP Isoforms in the Mouse Outer Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3720.
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© ARVO (1962-2015); The Authors (2016-present)
Information transfer in mammalian horizontal cells (HC) is poorly understood. The occurrence of clear–core vesicles, the vesicular GABA transporter, and several synaptic and vesicular proteins in HCs suggest transmitter release is mediated by a vesicular mechanism. In the mammalian presynaptic nerve terminal, formation of a complex between VAMP1 or VAMP2, syntaxin1 and SNAP–25 is known to mediate fusion of the vesicle and target membranes. To determine whether the molecular machinery for vesicular transmitter release is present in HCs, we examined the distribution of one component of this complex, the vesicle–associated membrane proteins (VAMP), in the outer retina.
Immunohistochemistry with antibodies (AB) to all 7 known isoforms of VAMPs, and to the VAMP–like proteins tomosyn and amisyn were used to evaluate mouse outer retina. Double labelings were performed with ABs to calbindin D–28k, a HC marker, and to vesicular glutamate transporter 1 (VGLUT1) or PSD95 to identify photoreceptor (PR) terminals. Immunostaining was evaluated using confocal microscopy.
Strong VAMP1 immunoreactive puncta embedded in the synaptic triad of the PR terminals were observed in the outer plexiform layer (OPL) using a VAMP1–specific AB (ABCAM). Double label studies showed that these puncta were in the tips of HC dendrites and axons, although we could not eliminate the possible expression of VAMP1 in PR synaptic ribbons due to their proximity. In contrast, strong immunoreactivity was in PR terminals using a second VAMP1 AB (Synaptic Systems), presumably recognizing VAMP1 and VAMP2, and two different VAMP2 ABs. The presence of VAMP1 or VAMP2 immunoreactivity in HC tips could not be eliminated because they are deeply embedded in PR terminals and immunoreactivity was distributed across the entire terminal. Weak VAMP4 immunoreactivity (ABCAM), compared to VAMP1 and VAMP2, was also in PR terminals. Moreover, PR terminal immunostaining was absent using a second VAMP4 AB (Synaptic Systems). VAMP3 immunoreactivity was in bipolar cell dendrites, and weak tomosyn immunoreactivity was in the OPL, although it was not possible to distinguish if tomosyn was colocalized with HC tips or PR terminals. No immunostaining was observed in the OPL using VAMP5, VAMP7, VAMP8 and amisyn ABs.
VAMP1 and tomosyn are promising candidates for participating in vesicular transmitter release from HCs. However, further characterizations using single cell RT–PCR and ultrastructural studies are necessary to definitely establish the molecular identity and cellular distribution of the VAMP and VAMP–like isoforms expressed in HCs.
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